Neal L. Millar

MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis

MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14+ cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68+ cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14+ cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14+ cells reduced TNF-α production. Finally, miR-155–deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.

Cytokines and apoptosis in supraspinatus tendinopathy

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

Inflammation is Present in Early Human Tendinopathy

Background The cellular mechanisms of tendinopathy remain unclear particularly with respect to the role of inflammation in early disease. The authors previously identified increased levels of inflammatory cytokines in an early human model of tendinopathy and sought to extend these studies to the cellular analysis of tissue. Purpose To characterize inflammatory cell subtypes in early human tendinopathy, the authors explored the phenotype and quantification of inflammatory cells in torn and control tendon samples. Design Controlled laboratory study. Methods Torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from 20 patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery. Tendon biopsy samples were evaluated immunohistochemically by quantifying the presence of macrophages (CD68 and CD206), T cells (CD3), mast cells (mast cell tryptase), and vascular endothelium (CD34). Results Subscapularis tendon samples obtained from patients with a torn supraspinatus tendon exhibited significantly greater macrophage, mast cell, and T-cell expression compared with either torn supraspinatus samples or control subscapularis-derived tissue (P < .01). Inflammatory cell infiltrate correlated inversely (r = .5; P < .01) with rotator cuff tear size, with larger tears correlating with a marked reduction in all cell lineages. There was a modest but significant correlation between mast cells and CD34 expression (r = .4; P < .01) in matched subscapularis tendons from shoulders with supraspinatus ruptures. Conclusion This study provides evidence for an inflammatory cell infiltrate in early mild/moderate human tendinopathy. In particular, the authors demonstrate significant infiltration of mast cells and macrophages, suggesting a role for innate immune pathways in the events that mediate early tendinopathy. Clinical Relevance Further mechanistic studies to evaluate the net contribution and hence therapeutic utility of these cellular lineages and their downstream processes may reveal novel therapeutic approaches to the management of early tendinopathy.

IL-33 induces skin inflammation with mast cell and neutrophil activation

Psoriasis is a common chronic autoimmune condition of the skin characterized by hyperplasia of epidermal keratinocytes associated with pro‐inflammatory cytokines. IL‐33 is a new member of the IL‐1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently, broader immune activatory potential has been defined for IL‐33 particularly via mast cell activation and neutrophil migration. Here, we show that ST2−/− mice exhibit reduced cutaneous inflammatory responses compared with WT mice in a phorbol ester‐induced model of skin inflammation. Furthermore, injections of IL‐33 into the ears of mice induce an inflammatory skin lesion. This inflammatory response was partially dependent on mast cells as mast cell‐deficient mice (KitW‐sh/W‐sh) showed delayed responses to IL‐33. IL‐33 also recruited neutrophils to the ear, an effect mediated in part by increased production of the chemokine KC (CXCL1). Finally, we show that IL‐33 expression is up‐regulated in the epidermis of clinical psoriatic lesions, compared with healthy skin. These results therefore demonstrate that IL‐33 may play a role in psoriasis‐like plaque inflammation. IL‐33 targeting may provide a new treatment strategy for psoriasis.

IL-33 Shifts the Balance from Osteoclast to Alternatively Activated Macrophage Differentiation and Protects from TNF-α–Mediated Bone Loss

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2−/− bone marrow led to more bone loss compared with the chimeras with ST2+/+ bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206+ AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Hypoxia: a critical regulator of early human tendinopathy

Objectives To seek evidence for the role of hypoxia in early human tendinopathy, and thereafter to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediator expression and matrix regulation in human tenocytes. Methods Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of the subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative real-time PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V fluorescence-activated cell sorter staining. Results Increased expression of hypoxia-inducible factor 1α, Bcl-2 and clusterin was detected in subscapularis tendon samples compared with both matched torn samples and non-matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of collagen type III operating through a mitogen-activated protein kinase-dependent pathway. Finally, hypoxia increased the expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Conclusion Hypoxia promotes the expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. The authors propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders.

Heat Shock Protein and Apoptosis in Supraspinatus Tendinopathy

Heat shock proteins (HSPs) are often upregulated following oxidative and other forms of stress. Based on reports of excessive apoptosis in torn supraspinatus tendon and mechanically loaded tendon cells, we hypothesized heat shock proteins may be present in rodent and human models of tendinopathy due to their central role in caspase dependent apoptotic cell signaling. We used a running rat supraspinatus tendinopathy overuse model with custom microarrays to investigate the process at a genetic level. Additionally torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. We identified substantial upregulation of heat shock proteins and apoptotic genes in the rodent model. We further confirmed increased levels of heat shock protein and apoptotic regulatory genes in human supraspinatus and subscapularis tendon. This finding suggests heat shock proteins play a role in the cascade of stress-activated programmed cell death and degeneration in tendinopathy and may provide a novel target in preventing tendinopathies.

MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease.

MicroRNA (miRNA) has the potential for cross-regulation and functional integration of discrete biological processes during complex physiological events. Utilizing the common human condition tendinopathy as a model system to explore the cross-regulation of immediate inflammation and matrix synthesis by miRNA we observed that elevated IL-33 expression is a characteristic of early tendinopathy. Using in vitro tenocyte cultures and in vivo models of tendon damage, we demonstrate that such IL-33 expression plays a pivotal role in the transition from type 1 to type 3 collagen (Col3) synthesis and thus early tendon remodelling. Both IL-33 effector function, via its decoy receptor sST2, and Col3 synthesis are regulated by miRNA29a. Downregulation of miRNA29a in human tenocytes is sufficient to induce an increase in Col3 expression. These data provide a molecular mechanism of miRNA-mediated integration of the early pathophysiologic events that facilitate tissue remodelling in human tendon after injury.

Inflammatory mechanisms in tendinopathy - towards translation

Tendinopathy is a multifactorial spectrum of tendon disorders that affects different anatomical sites and is characterized by activity-related tendon pain. These disorders are common, account for a high proportion (∼30%) of referrals to musculoskeletal practitioners and confer a large socioeconomic burden of disease. Our incomplete understanding of the mechanisms underpinning tendon pathophysiology continues to hamper the development of targeted therapies, which have been successful in other areas of musculoskeletal medicine. Debate remains among clinicians about the role of an inflammatory process in tendinopathy owing to a lack of clinical correlation. The advent of modern molecular techniques has highlighted the presence of immune cells and inflammatory mechanisms throughout the spectrum of tendinopathy in both animal and human models of disease. Key inflammatory mediators — such as cytokines, nitric oxide, prostaglandins and lipoxins — play crucial parts in modulating changes in the extracellular matrix within tendinopathy. Understanding the links between inflammatory mechanisms, tendon homeostasis and resolution of tendon damage will be crucial in developing novel therapeutics for human tendon disease.

The effectiveness of arthroscopic stabilisation for failed open shoulder instability surgery

We identified ten patients who underwent arthroscopic revision of anterior shoulder stabilisation between 1999 and 2005. Their results were compared with 15 patients, matched for age and gender, who had a primary arthroscopic stabilisation during the same period. At a mean follow-up of 37 and 36 months, respectively, the scores for pain and shoulder function improved significantly between the pre-operative and follow-up visits in both groups (p = 0.002), with no significant difference between them (p = 0.4). The UCLA and Rowe shoulder scores improved significantly (p = 0.004 and p = 0.002, respectively), with no statistically significant differences between groups (p = 0.6). Kaplan-Meier analysis for time to recurrent instability showed no differences between the groups (p = 0.2). These results suggest that arthroscopic revision anterior shoulder stabilisation is as reliable as primary arthroscopic stabilisation for patients who have had previous open surgery for recurrent anterior instability.